Novel functional peptide nucleic acid monomer and process for producing the same

ABSTRACT

A compound represented by general formula (I) below;  
                 
 
     (in the formula, A denotes  
                 

                 

                 

                 
 
     B denotes  
                 
 
     R denotes H, NO 2 , NH 2 , NHCbz, Br, F, Cl or SO 3 Na 2 , and n is an integer of 1 to 4), and a process for producing the above compound characterised in that it includes a reaction between an activated ester and a t-butoxycarbonylaminoethylamine or an ω-amino acid derivative.

TECHNICAL FIELD

[0001] The present invention relates to a functional peptide nucleicacid monomer having a novel structure and a production process therefor.

BACKGROUND ART

[0002] Nucleic acids are the DNA and RNA that govern the geneticinformation of living creatures. On the other hand, a peptide nucleicacid (PNA) is a modified nucleic acid in which the sugar-phosphateskeleton of a nucleic acid has been converted into anN-(2-aminoethyl)glycine skeleton (FIG. 1). The sugar-phosphate skeletonsof DNA/RNA are negatively charged under neutral conditions and exhibitelectrostatic repulsion between the complementary strands, but since thebackbone structure of PNA itself has no charge, there is noelectrostatic repulsion. PNA therefore has a high duplex-forming abilityand a high base sequence recognition ability in comparison withconventional nucleic acids. Furthermore, since PNA is very stableagainst in vivo nuclease/protease and is not decomposed thereby, itsapplication in gene therapy as an antisense molecule has beeninvestigated.

[0003] Modifying conventional techniques that employ DNA as a medium sothat they can be used with PNA can compensate for the defects of DNAthat could not be overcome previously. For example, it is possible toapply PNA to the “DNA microarray technology” that carries out asystematic analysis of a large amount of genetic information at highspeed, and to the “molecular beacon” that has been developed recently asa probe that can detect by fluorescence a specifically recognised basesequence. Since these techniques use DNA as a medium, which has poorenzyme resistance, when employing these techniques it is necessary tocarry out precise sampling. Satisfying this requirement is the key toenhancing the above-mentioned techniques.

[0004] On the other hand, since PNA is completely resistant to enzymes,the use of PNA as a replacement for DNA in the DNA microarray technologyand the molecular beacon is anticipated to eliminate the defects of theabove-mentioned techniques and to derive further advantages.

[0005] There are a large number of fields, in addition to the DNAmicroarray technology and the molecular beacon, that are anticipated toadvance as a result of the use of PNA, and in these fields it isnecessary to efficiently functionalise PNA, that is to say, to design anovel PNA monomer by the efficient introduction of a functional moleculeto a PNA monomer.

[0006] Since methods for synthesising a PNA oligomer employ the commonlyused solid phase peptide synthesis, PNA monomer units can be classifiedinto two types in terms of the PNA backbone structure, that is to say,the Fmoc type PNA monomer unit and the tBoc type PNA monomer unit (FIG.2).

[0007] A method for synthesising the Fmoc type PNA monomer unit hasalready been established, its oligomers can be synthesised by means of astandard automatic DNA synthesiser, and it can be synthesised on a smallscale by the route below:

[0008] (X denotes guanine, thymine, cytosine or adenine.)

[0009] The first PNA employed the tBoc type PNA monomer unit asdescribed below:

[0010] after which a more efficient synthetic method:

[0011] was established.

[0012] However, as described above, since the Fmoc type, which is easyto handle, has been developed, the use of the tBoc type is becoming lessfrequent.

[0013] However, when introducing a functional molecule other than thefour nucleic acids guanine, thymine, cytosine and adenine, for example,when introducing a photofunctional molecule, since the functionalmolecule that is to be introduced is often unstable under alkalineconditions, the tBoc type PNA backbone structure, which does not employalkaline conditions, is very useful. With regard to a “process forproducing t-butoxycarbonylaminoethylamines and amino acid derivatives”,there is already a patent application filed by the present inventors asJapanese Patent Application No. 2000-268638.

[0014] Other than the above process, 5 examples of the synthesis of amonomer unit for a photofunctional PNA oligomer are known. All thesecases employ the above-mentioned route, but their yields are either notdescribed or very low (Peter E. Nielsen, Gerald Haaiman, Anne B. EldrupPCT Int. Appl. (1998) WO 985295 A1 19981126, T. A. Tran, R. -H. Mattern,B. A. Morgan (1999) J. Pept. Res, 53, 134-145, Jesper Lohse et al.(1997) Bioconjugate Chem., 8, 503-509, Hans-georg Batz, HenrikFrydenlund Hansen, et al. PCT Int. Appl. (1998) WO 9837232 A2 19980827,Bruce Armitage, Troels Koch, et al. (1998) Nucleic Acid Res., 26,715-720, Hans-georg Batz, Henrik Frydenlund Hansen, et al.) Furthermore,since the structures of the compounds used have the characteristic ofbeing comparatively stable under alkaline conditions, it is surmisedthat, when a chromophore that is unstable under alkaline conditions ispresent, an efficient synthesis by a method similar to theabove-mentioned conventional method, that is to say, route A below, isnot possible.

[0015] There is therefore a strong desire for the establishment of atechnique that functionalises a PNA monomer efficiently as well as forthe development of a functional PNA monomer such as, for example, aphotofunctional PNA monomer.

DISCLOSURE OF INVENTION

[0016] It is an object of the present invention to provide a novelfunctional PNA monomer and an efficient synthetic method therefor thatcan eliminate the above-mentioned problems.

[0017] As a result of an intensive investigation by the presentinventors in order to solve the above-mentioned problems, as shown inroute B below, a photofunctional PNA monomer 4 has been synthesisedsuccessfully and substantially stoichiometrically using at-butoxycarbonylaminoethylamine derivative 6 as a PNA backbone structureby condensation with an activated ester derivative 5 containing apentafluorophenyl group 1.

[0018] Furthermore, the present inventors have also succeeded insynthesising a photofunctional PNA monomer 4 substantiallystoichiometrically using an ω-amino acid derivative 8 as a PNA backbonestructure by condensation with an activated ester derivative 7containing a pentafluorophenyl group 1 as shown in route C below:

[0019] The present inventors have found that the above-mentionedproblems can be solved by the above-mentioned routes B and C, and thepresent invention has thus been accomplished.

[0020] That is to say, the present invention relates to a compoundrepresented by general formula (I) below.

[0021] (In the formula, A denotes

[0022] R denotes H, NO₂, NH₂, NHCbz, Br, F, Cl or SO₃Na₂, and n is aninteger of 1 to 4, wherein when A denotes

[0023] then B denotes

[0024] Furthermore, the present invention relates to a process forproducing a functional PNA monomer by reacting at-butoxycarbonylaminoethylamine with a derivative of a functionalmolecule so as to incorporate the functional molecule into a PNAmonomer, characterised in that the derivative of a functional moleculeis an activated ester.

[0025] Furthermore, the present invention relates to the above-mentionedproduction process, characterised in that the activated ester has, onthe carbonyl carbon forming the ester bond, a group represented bygeneral formula (II) below:

[0026] (In the formula, A denotes

[0027] R denotes H, NO₂, NH₂, NHCbz, Br, F, Cl or SO₃Na₂, and n is aninteger of 1 to 4.).

[0028] Furthermore, the present invention relates to the above-mentionedproduction process, characterised in that the activated ester has, onits carbonyl carbon, a pentafluorophenoxy group or a succinimidoxygroup.

[0029] Furthermore, the present invention relates to a process forproducing an activated ester, characterised in that it includes reactinga carboxylic acid derivative of a functional molecule with a compoundhaving a pentafluorophenoxy group or a succinimidoxy group.

[0030] Furthermore, the present invention relates to a process forproducing a carboxylic acid derivative of a functional molecule,characterised in that it includes reacting a derivative of thefunctional molecule with an aliphatic carboxylic acid.

[0031] Furthermore, the present invention relates to a process forproducing a functional PNA monomer from a functional molecule,including:

[0032] producing a derivative of the functional molecule from thefunctional molecule,

[0033] producing a carboxylic acid derivative of the functional moleculefrom the derivative of the functional molecule,

[0034] producing an activated ester from the carboxylic acid derivativeof the functional molecule, and

[0035] producing a functional PNA monomer from the activated ester,characterised in that it includes one or more of a) to c) below:

[0036] a) in the production of the carboxylic acid derivative of thefunctional molecule, reacting the derivative of the functional moleculewith an aliphatic carboxylic acid;

[0037] b) in the production of the activated ester, reacting thecarboxylic acid derivative of the functional molecule with a compoundhaving a pentafluorophenoxy group or a succinimidoxy group; and

[0038] c) in the production of the functional PNA monomer, reacting at-butoxycarbonylaminoethylamine with the activated ester derivative ofthe functional molecule.

[0039] Furthermore, the present invention relates to a process forproducing a functional PNA monomer by reacting a derivative of afunctional molecule with an ω-amino acid derivative represented bygeneral formula (III) below, thereby incorporating the functionalmolecule into a PNA monomer, characterised in that the derivative of thefunctional molecule is an activated ester:

[0040] (In the formula, R¹ denotes a hydrogen atom or a straight- orbranched-chain C₁ to C₅ alkyl group, and m denotes an integer of 1 to11.).

[0041] Furthermore, the present invention relates to the above-mentionedproduction process, characterised in that the activated ester has, onits carbonyl group forming an ester bond, a group represented by generalformula (II) below, either directly or via an aliphatic chain or apeptide chain:

[0042] (In the formula, A denotes

[0043] R denotes H, NO₂, NH₂, NHCbz, Br, F, Cl or SO₃Na₂, and n is aninteger of 1 to 4:).

[0044] Furthermore, the present invention relates to the above-mentionedproduction process, characterised in that the activated ester has, onits carbonyl carbon, a pentafluorophenoxy group or a succinimidoxygroup.

[0045] Furthermore, the present invention relates to a process forproducing a functional PNA monomer from a functional molecule, includingproducing an activated ester from the functional molecule and producinga functional PNA monomer from the activated ester, characterised in that

[0046] the production of the activated ester from the functionalmolecule includes reacting m-methyl red with a compound that contains asuccinimidoxy group, and/or

[0047] the production of the functional PNA monomer from the activatedester includes reacting a benzyloxycarbonyl-ω-amino acid derivativerepresented by general formula (III) with the activated ester derivativeof the functional molecule, thereby incorporating the functionalmolecule into a PNA monomer.

[0048] Features of the present invention are now explained in detail bycomparing the processes of the present invention with the conventionalprocesses.

[0049] The synthesis of tBoc type PNA monomer unit 4 usually employsroute A below:

[0050] That is to say, it is a process in which, after synthesising 3 bydehydration condensation between 1 and 2, 3 is subjected to alkalinehydrolysis to give 4. This process is also used when introducing thefour nucleic acid base groups guanine, thymine, cytosine, and adenine.When introducing a functional molecule (this refers to a known compoundcited in Table 1) other than the above-mentioned groups, route A is alsoemployed. However, since many photofunctional molecules are generallyunstable under alkaline conditions, it is impossible to obtain 4efficiently by route A. Instead of using the conventional PNA backbonestructure 2, the condensation is therefore carried out using 6 obtainedby the prior hydrolysis of 2. Since 6 includes a free carboxylic acidgroup, as is the case for 1, there is a possibility that anintramolecular dehydration condensation might occur, and a directdehydration condensation using a condensing agent such as DCC cannottherefore be employed. The reaction is therefore devised so that thefree carboxylic acid group and secondary amino group of 6 do notcondense intramolecularly with each other by converting 1 into anactivated ester derivative 5 using pentafluorophenol and then reacting 5with 6 (route B). This gives 4 stoichiometrically. As hereinbeforedescribed, there is no precedent for the process of synthesising 4 byconverting a photofunctional molecule into its activated ester and thenreacting it with 6, and it can be said that this process will hereafterbe an indispensable technique for the synthesis of a variety ofphotofunctional PNA monomers.

[0051] Since benzyloxycarbonyl-ω-amino acid derivatives represented bygeneral formula (III) below, which are used in route C, already have alinker (carboxylamino acid):

[0052] (in the formula, R¹ denotes a hydrogen atom or a straight- orbranched-chain C₁ to C₅ alkyl group, and m denotes an integer of 1 to11),

[0053] the derivatives are very versatile, and reaction of thederivatives with an activated ester derivative gives a target functionalPNA monomer unit in one step. Moreover, many commercially availablebenzyloxycarbonyl-ω-amino acid derivatives can be used. The route C istherefore particularly effective when targeting a comparativelyexpensive photofunctional molecule.

[0054] On the other hand, incorporation of a photofunctional moleculesuch as a sulphonyl chloride type or the highly sterically hinderedmethyl red into a PNA monomer can be carried out desirably by route B.

[0055] It is therefore possible to synthesise a variety of functionalPNA monomers by appropriately choosing one from the synthetic method viaroute B and that via route C of the present invention.

[0056] In accordance with the present invention, photofunctional monomerunits such as a Naphthalimide type, a Flavin type, a Dabcyl type, aBiotin type, an FAM type, a Rhodamine type, a TAMRA type, an ROX type,an HABA type, a Pyrene type and a Coumarin type can be obtained. It isalso possible to obtain photofunctional monomer units other than theabove-mentioned types and to obtain functional monomer units other thanthe photofunctional monomer units.

BRIEF DESCRIPTION OF DRAWINGS

[0057]FIG. 1 is a diagram showing differences in the structure and theelectrical charge between DNA and PNA.

[0058]FIG. 2 is a diagram showing the structures of two types of PNAmonomer unit.

MODES FOR CARRYING OUT THE INVENTION

[0059] Conventionally, in the synthesis of a photofunctional monomerunit, for example, in the synthesis of compound 4a,

[0060] a carboxylic acid derivative 1a of a functional molecule and aPNA backbone structure 2a are subjected to dehydration condensation togive 3a, which is then subjected to alkaline hydrolysis to give thetarget 4a (route A). Although the flavin skeleton of 1a is stabletowards acid, it is easily decomposed under alkaline conditions to give6,7-dimethylquinoxalinedione, and it is therefore impossible to obtain4a efficiently even when 3a can be synthesised. When 1a is convertedinto an activated ester derivative 5a and then reacted with 6a, thereaction proceeds substantially stoichiometrically to give 4a with ayield of 85% (route B).

[0061] In the synthesis of compound 4b,

[0062] although a naphthalimide derivative 3b is obtained from thecorresponding 1b and 2b with a yield of 44% the subsequent alkalinehydrolysis gives 4b in only 4% (route A). When 1b is converted intoactivated ester derivative 5b and then reacted with 6b, the reactionproceeds to give 4b with a yield of 75% (route B).

[0063] The production of 1a and 1b, which are carboxylic acidderivatives, is carried out using an aliphatic carboxylic acid, andpreferable a straight-chain aliphatic carboxylic acid.

[0064] In the production of a monomer that is incorporated at a terminalamino group of a PNA, an activated ester having a succinimide group canpreferably be used.

[0065] As the ω-amino acid derivative represented by general formula(III) that is used in route C, one having a C₁ to C₁₁ carbon chain onthe carbonyl carbon of its amino acid moiety can be used, but since aPNA is generally expected to be a hybrid with DNA, it is desirable forthe derivative to be similar sterically to DNA. Taking this point intoconsideration, when a carboxylamino acid is used as a linker, one havingZ-glycine as an amino moiety is the most suitable.

[0066] With regard to the method for synthesising a photofunctional PNAoligomer, the Fmoc method and the tBoc method can be used. The Fmocmethod involves a protecting group removal step using an alkalinereagent, and its use is inappropriate when designing a photofunctionalPNA oligomer. On the other hand, since the tBoc method does not employalkaline conditions in its synthetic steps, it is suitable as asynthetic method for a photofunctional PNA oligomer. Application of thePNA monomer related to the present invention to the tBoc methodtherefore allows a photofunctional PNA oligomer to be synthesisedefficiently.

EXAMPLES

[0067] The present invention is explained below in more detail by way ofexamples, but the present invention is in no way limited thereby.

Example 1 Synthesis of 2,3,4,5,6-pentafluorophenyl2-(5,7,8trimethyl-1,3-dioxo-2,5-dihydro-2,4-diazaphenazin-2-yl)acetate(5a)

[0068] EDC (73.2 mg, 382 μmol) was added to a DMF solution (10 ml) of 1a(100 mg, 318 μmol) and PfpOH (70.2 mg, 381 μmol) at 0° C., and thisreaction mixture was stirred at 0° C. for 1 hour and at room temperaturefor 12 hours. This reaction mixture was concentrated under reducedpressure, and the residue was subjected to partition extraction with awater-chloroform system. The organic layer was dried with magnesiumsulphate and concentrated under reduced pressure, and the residue wasthen purified by silica gel column chromatography (2.5% MeOH/CHCl₃) togive 5a (130 mg, 85%). ¹H NMR (CDCl₃) δ 8.07 (s, 1 H), 7.44 (s, 1 H),5.21 (s, 2 H), 4.14 (s, 3 H), 2.55 (s, 3 H), 2.45 (s, 3 H); HRMS (FAB⁺,NBA/CH₂Cl₂) C₂₁H₁₄O₄N₄F₅ [(M+H)⁺] Calcd. 481.0934, Exp. 481.0950; UVλmax (DMF) 390, 460 (nm).

Example 2 Synthesis of2-(N-(2-((t-butoxy)carbonylamino)ethyl))-2-(5,7,8-trimethyl-1,3-dioxo(2,5-dihydro-2,4-diazaphenazin-2yl)acetylamino)acetic acid (4a)

[0069] Diisopropylethylamine (36.3 μL, 208 μmol) was added to a DMFsolution (10 mL) of 5a (100 mg, 208 μmol) and 6 (45.4 mg, 208 μmol) andstirred at room temperature for 15 hours. This was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (10-50% MeOH/CHCl₃) to give 4a (130 mg, 85%). ¹H NMR(CD₃OD) δ 7.94 (s) and 7:86 (s) (1 H), 7.80 (s) and 7.75 (s) (1 H), 5.03(s) and 4.88 (s) (2 H), 4.17 (s) and 4.13 (s) (3 H), 3.64 and 3.52 (2H), 3.38 and 3.26 (2 H), 2.58 (s) and 2.56 (s) (3 H), 2.46 (s) and 2.44(s) (3 H), 1.46 (s) and 1.41 (s) (9 H); HRMS (FAB⁺, NBA/CH₂Cl₂)C₂₄H₃₁O₇N₆[(M+H)⁺] Calcd. 515.2252, Exp. 515.2273; UV λmax (DMF) 390,460 (nm).

Example 3 Synthesis of N-(4-dimethylaminoazobenzene-2′-carbonyl)glycine(1c)

[0070] Triethylamine (732 μL, 5.25 mmol) was added to a DMF solution (10mL) of methyl red (1.35 g, 5 mmol) and t-butylglycine hydrochloride (880mg, 5.25 mmol), while ice cooling DCC (1.13g, 5.5 mmol) was addedthereto and this was stirred for 30 minutes and further at roomtemperature for 15 hours. The reaction mixture was filtered, thefiltrate was concentrated under reduced pressure, and the residue waspurified by silica gel column chromatography (0-10% acetone/CH₂Cl₂) togive the t-butyl ester derivative (1.05 g, 55%) of 1c as orangeneedle-shaped crystals. This derivative (765 mg, 2 mmol) was added toformic acid (50 mL), stirred at room temperature for 2 days, andconcentrated under reduced pressure to remove the formic acid, and theresidue was purified by silica gel column chromatography (0-5%acetone/CH₂Cl₂) to give 1c (549 mg, 84%) as red needle-shaped crystals.¹H NMR (CDCl₃) δ 9.99 (brt, 1H), 8.40 (d, J=8 Hz, 1 H), 7.89 (d, J=9 Hz,2 H), 7.84 (d, J=8 Hz, 1H), 7.56 (t, J=8 Hz, 1 H), 7.49 (t, J=8 Hz, 1H), 6.75 (d, J=9 Hz, 2H), 4.42 (d, J=5 Hz, 2 H), 3.10 (s, 6 H); ¹³C NMR(CDCl₃) δ 167.61; 153.39, 150.86, 143.30, 132.49, 131.47, 129.38,127.69, 126.28, 116.24, 111.63, 43.20, 40.24;

Example 4 Synthesis of 2,3,4,5,6-pentafluorophenylN-(4-dimethylaminoazobenzene-2′-carbonyl)glycinate (5c)

[0071] DCC (308 mg, 1.5 mmol) was added to a DMF solution (10 mL) of 1c(326 mg, 1 mmol) and PfpOH(276 mg, 1.5 mmol) while ice cooling, and thisreaction mixture was stirred at room temperature for 15 hours. Thereaction mixture was filtered, the filtrate was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (0-5% acetone/CH₂Cl₂) to give 5c (449 mg, 91%) as anorange powder. 1H NMR (CDCl₃) δ 10.14 (brt, 1 H), 8.37 (d, J=8 Hz, 1 H),7.78 (d, J=9 Hz, 2 H), 7.76 (d, J=8 Hz, 1 H), 7.57 (t, J=8 Hz, 1 H),7.50 (t, J=8 Hz, 1 H), 6.74 (d, J=9 Hz, 2 H), 4.68 (d, J=5 Hz, 2 H) 3.06(s, 6 H).

Example 5 Synthesis of2-(N-(2-((t-butoxy)carbonylamino)ethyl)-2-(4-dimethylaminoazobenzene-2′-carbonylamino)acetylamino)aceticacid (4c)

[0072] Diisopropylethylamine (85 μL, 0.5 mmol) was added to a DMFsolution (5 mL) of 5c (246 mg, 0.5 mmol) and 6 (109 mg, 0.5 mmol) andstirred at room temperature for 15 hours. This was concentrated underreduced pressure and the residue was purified by silica gel columnchromatography (0-30% MeOH/CH₂Cl₂) to give 4c (225 mg, 72%). ¹H NMR(CDCl₃) δ 9.99 (s) and 9.85 (s) (1 H), 8.3-7.6 (m, 4 H), 7.4-7.2 (m, 2H), 6.67 (s) and 6.59 (s) (2 H), 5.62 (s) and 5.27 (s) (1 H), 4.35 (s)and 4.20 (s) (2 H), 3.99 and 3.90 (2 H), 3.5 (brs) and 3.3 (brs) (2 H),3.2 (brs) and 3.0 (brs) (2 H), 2.99 (s) and 2.87 (s) (6 H), 1.25 (brs, 9H).

[0073] The route from 1c to 4c was as follows:

Example 6 Synthesis of N-(4-hydroxyazobenzene-2-carbonyl)glycine (1d)

[0074] Triethylamine (732 μL, 5.25 mmol) was added to a DMF solution (10mL) of HABA (1.21 g, 5 mmol) and t-butylglycine hydrochloride (880 mg,5.25 mmol), while ice cooling DCC (1.13 g, 5.5 mmol) was then added andthis was stirred for 30 minutes and further at room temperature for 15hours. The reaction mixture was filtered, the filtrate was concentratedunder reduced pressure, and the residue was purified by silica gelcolumn chromatography (0-5% acetone/CH₂Cl₂) to give the t-butyl esterderivative (1.73 g, 97%) of 1d as an orange powder. This (1.07 g, 3mmol) was added to formic acid (50 mL), stirred at room temperature for2 days, and concentrated under reduced pressure to remove the formicacid, and the residue was purified by silica gel column chromatography(0-5% acetone/CH₂Cl₂) to give 1d (0.89 g, 99%) as an orange powder. ¹HNMR (CDCl₃) δ 10.45 (brt, 1 H), 8.88 (brt, J=5 Hz, 1 H), 7.91 (d, J=9Hz, 2 H), 7.85 (d, J=8 Hz, 1 H), 7.70 (d, J=8 Hz, 1 H), 7.60 (t, J=8 Hz,1 H), 7.57 (t, J=8 Hz, 1 H), 6.94 (d, J=9 Hz, 2 H), 4.05 (d, J=5 Hz, 2H); ¹³C NMR (CDCl₃) δ 171.18, 166.44, 161.54, 149.13, 145.34, 133.20,131.09, 130.18, 129.61, 125.86, 116.00, 115.88, 41.60.

Example 7 Synthesis of 2,3,4,5,6-pentafluorophenylN-(4-hydroxyazobenzene-2′-carbonyl)glycinate (5d)

[0075] DCC (308 mg, 1.5 mmol) was added to a DMF solution (10 mL) of 1d(299 mg, 1 mmol) and PfpOH (276 mg, 1.5 mmol) while ice cooling, andthis reaction mixture was stirred at room temperature for 15 hours. Thereaction mixture was filtered, the filtrate was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (0-5% acetone/CH₂Cl₂) to give 5d (46 mg, 10%) as anorange powder. ¹H NMR (CDCl₃) δ 9.67 (brs, 1 H), 9.02 (brt, 1 H),7.8-7.7 (m, 3 H), 7.6-7.5 (m, 2 H) 7.23 (d, J=9 Hz, 1H), 6.86 (d, J=9Hz, 2 H), 4.67 (d, J=5 Hz, 2 H).

Example 8 Synthesis of2-(N-(2-((t-butoxy)carbonylamino)ethyl)-2-(4hydroxyazobenzene-2′-carbonylamino)acetylamino)aceticacid (4d)

[0076] Diisopropylethylamine (14 μL, 80 μmol) was added to a DMFsolution (5 mL) of 5d (37 mg, 80 μmol) and 6 (18 mg, 80 μmol) andstirred at room temperature for 15 hours. This was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (0-30% MeOH/CH₂Cl₂) to give 4d (13 mg, 33%). ¹H NMR(CDCl₃) δ 9.77 (s) and 9.59 (s) (1 H), 8.26 (s) and 8.14 (s) (2H),7.9-7.6 (m, 2 H), 7.6-7.3 (m, 2 H), 7.0-6.6 (m, 2 H), 5.35 (s) and5.05(s) (1 H), 4.40 (s) and 4.24 (s) (2 H), 3.98 (s, 2 H), 3.6-3.3 (m, 2H), 3.21 (s) and 3.02 (s) (2 H), 1.28 (s) and 1.18 (s) (9 H).

[0077] The route from 1d to 4d was as follows.

Example 9 Synthesis of FAM-Gly-^(Boc)PNA-OH

[0078] 5,6-FAM N-hydroxysuccinimide ester (50 mg, 0.11 mmol) andtriethylamine (250 μL, 2.0 mmol) were added in that order to adimethylformamide solution (5 mL) of Gly-^(Boc)PNA-OH (30.3 mg, 0.10mmol) and stirred at room temperature for 15 hours. After completion ofthe reaction, the mixture was concentrated under reduced pressure, andthe residue was subjected to silica gel column chromatography (0-25%MeOH/dichloromethane) to give FAM-Gly-^(Boc)PNA-OH (69.8 mg, 100%) as ayellow powder. FABMS m/z 634 [(M+H)⁺]; HRMS (FAB⁺) calcd. forC₃₂H₃₂O₁₁N₃ [(M+H)⁺] 634.1959, observed 634.2034.

Example 10 Synthesis of TAMRA-Gly-^(Boc)PNA-OH

[0079] 5,6-TAMRA N-hydroxysuccinimide ester (5 mg, 9.5 μmol) andtriethylamine (20 μL, 140 μmol) were added in that order to adimethylformamide solution (5 mL) of Gly-^(Boc)PNA-OH (5.8 mg, 21 μmol)and stirred at room temperature for 15 hours. After completion of thereaction, the mixture was concentrated under reduced pressure, and theresidue was subjected to silica gel column chromatography (0-30%MeOH/dichloromethane) to give TAMRA-Gly-^(Boc)PNA-OH (6 mg, 100%) asreddish purple powder. FABMS mlz 688 [(M+H)⁺]; HRMS (FAB⁺) calcd. forC₃₆H₄₂O₉N₅ [(M+H)⁺] 688.2904, observed 688.2993.

Example 11 Synthesis of ROX-Gly-^(Boc)PNA-OH

[0080] 5,6-ROX N-hydroxysuccinimide ester (5 mg, 8 μmol) andtriethylamine (20 μL, 140 μmol) were added in that order to adimethylformamide solution (5 mL) of Gly-^(Boc)PNA-OH (4.9 mg, 18 μmol)and stirred at room temperature for 15 hours. After completion of thereaction, the mixture was concentrated under reduced pressure, and theresidue was subjected to silica gel column chromatography (0-30%MeOH/dichloromethane) to give ROX-Gly-^(Boc)PNA-OH (6 mg, 100%) as apurple powder. FABMS m/z 792 [(M+H)⁺]; HRMS (FAB⁺) calcd. for C₄₄H₅₀O₉N₅[(M+H)⁺] 792.3530, observed 792.3615.

Example 12 Synthesis of 2,3,4,5,6-pentafluorophenylN-(4-dimethylaminoazobenzene-2′-carbonyl) glycinate (o-MR-Gly-Opfp)

[0081] DCC (308 mg, 1.5 mmol) was added to a DMF solution (10 mL) ofo-MR-Gly-OH (326 mg, 1 mmol) and PfpOH (276 mg, 1.5 mmol) while icecooling, and the reaction mixture was stirred at room temperature for 15hours. The reaction mixture was filtered, the filtrate was concentratedunder reduced pressure and the residue was purified by silica gel columnchromatography (0-5% acetone/CH₂Cl₂) to give o-MR-Gly-OPfp (449 mg, 91%)as an orange powder. ¹H NMR (CDCl₃) δ 10.14 (brt, 1 H), 8.37 (d, J=8 Hz,1 H), 7.78 (d, J=9 Hz, 2 H), 7.76 (d, J=8 Hz, 1 H), 7.57 (t, J=8 Hz, 1H), 7.50 (t, J=8 Hz, 1 H), 6.74 (d, J=9 Hz, 2 H), 4.68 (d, J=5 Hz, 2 H),3.06 (s, 6 H).

Example 13 Synthesis of2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-2-(4-dimethylaminoazobenzene-2′-carbonylamino)acetylamino)aceticacid (o-MR-Gly-^(Boc)PNA-OH)

[0082] Diisopropylethylamine (85 μL, 0.5 mmol) was added to a DMFsolution (5 mL) of o-MR-Gly-OPfp (246 mg, 0.5 mmol) and ^(Boc)PNA-OH(109 mg, 0.5 mmol) and stirred at room temperature for 15 hours. Thiswas concentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (0-30% MeOH/CH₂Cl₂) to giveo-MR-Gly-^(Boc)PNA-OH (225 mg, 72%). ¹H NMR (CDCl₃) δ 9.99 (s) and 9.85(s) (1 H), 8.3-7.6 (m) (4 H), 7.4-7.2 (m) (2 H), 6.67 (s) and 6.59 (s)(2 H), 5.62 (s) and 5.27 (s) (1 H), 4.35 (s) and 4.20 (s) (2 H), 3.99and 3.90 (2 H), 3.5 (brs) and 3.3 (brs) (2 H), 3.2 (brs) and 3.0 (brs)(2 H), 2.99 (s) and 2.87 (s) (6 H), 1.25 (brs, 9 H).

Example 14 Synthesis of 2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-2(4-dimethylaminoazobenzene-4′-carbonylamino)acetylamino)aceticacid (Dabcyl-Gly-^(Boc)PNA-OH (p-MR-Gly-^(Boc)PNA-OH))

[0083] Dabcyl N-hydroxysuccinimide ester (145 mg, 0.40 mmol) andtriethylamine (600 μL, 4.5 mmol) were added in that order to adimethylformamide solution (10 mL) of Gly-^(Boc)PNA-OH (100 mg, 0.39mmol) and stirred at room temperature for 15 hours. After completion ofthe reaction, the mixture was concentrated under reduced pressure, andthe residue was purified by silica gel column chromatography (0-4%MeOH/dichloromethane) to give Dabcyl-Gly-^(Boc)PNA-OH (184 mg, 90%) as areddish brown powder. ¹H NMR (DMSO-d₆) δ 8.18 (d, J=7 Hz, 2 H), 7.91 (d,J=7 Hz, 2 H), 7.88 (d, J=7 Hz, 2 H), 6.77 (d, J=7 Hz, 2 H), 5.76 (s) and5.30 (s) (2 H), 4.22 (brs) and 4.05 (brs) (2 H), 3.73 (brs) and 3.49(brs) (2 H), 3.47 (brs) and 3.29 (brs) (2 H), 1.26 (s, 9 H); FABMS m/z527 [(M+H)⁺].

Example 15 Synthesis of N-(4-hydroxyazobenzene-2′-carbonyl)glycine(HABA-Gly-OH)

[0084] Triethylamine (732 μL, 5.25 mmol) was added to a DMF solution (10mL) of HABA (1.21 g, 5 mmol) and t-butylglycine hydrochloride (880 mg,5.25 mmol), while ice cooling DCC (1.13 g, 5.5 mmol) was then addedthereto and the mixture was stirred for 30 minutes and further at roomtemperature for 15 hours. The reaction mixture was filtered, thefiltrate was concentrated under reduced pressure, and the residue waspurified by silica gel column chromatography (0-5% Acetone/CH₂Cl₂) togive the t-butyl ester derivative of HABA-Gly-OH (1.73 g, 97%) as anorange powder. This (1.07 g, 3 mmol) was added to formic acid (50 mL)and stirred at room temperature for 2 days, concentrated under reducedpressure to remove the formic acid, and the residue was purified bysilica gel column chromatography (0-5% acetone/CH₂Cl₂) to giveHABA-Gly-OH (0.89 g, 99%) as an orange powder. ¹H NMR (CDCl₃) δ 10.45(brt, 1 H), 8.88 (brt, J=5 Hz, 1 H), 7.91 (d, J=9 Hz, 2 H), 7.85 (d, J=8Hz, 1 H), 7.70 (d, J=8 Hz, 1 H), 7.60 (t, J=8 Hz, 1 H), 7.57 (t, J=8 Hz,1 H), 6.94 (d, J=9 Hz, 2 H), 4.05 (d, J=5 Hz, 2 H); ¹³C NMR (CDCl₃) δ171.18, 166.44, 161.54, 149.13, 145.34, 133.20, 131.09, 130.18, 129.61,125.86, 116.00, 115.88, 41.60.

Example 16 Synthesis of 2,3,4,5,6-pentafluorophenylN-(4-hydroxyazobenzene-2′-carbonyl)glycinate (HABA-Gly-Opfp)

[0085] DCC (308 mg, 1.5 mmol) was added to a DMF solution (10 mL) ofHABA-Gly-OH (299 mg, 1 mmol) and PfpOH (276 mg, 1.5 mmol) while icecooling, and the reaction mixture was stirred at room temperature for 15hours. The reaction mixture was filtered, the filtrate was concentratedunder reduced pressure, and the residue was purified by silica gelcolumn chromatography (0-5% acetone/CH₂Cl₂) to give HABA-Gly-OPfp (46mg, 10%) as an orange powder. ¹H NMR (CDCl₃) δ 9.67 (brs, 1 H), 9.02(brt, 1 H), 7.8-7.7 (m, 3 H), 7.6-7.5 (m, 2 H), 7.23 (d, J=9 Hz, 1 H),6.86 (d, J=9 Hz, 2 H), 4.67 (d, J=5 Hz, 2 H).

Example 17 Synthesis of2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-2-(4-hydroxyazobenzene-2′-carbonylamino)acetylamino)aceticacid (HABA-Gly-^(Boc)PNA-OH)

[0086] Diisopropylethylamine (14 mL, 80 μmol) was added to a DMFsolution (5 mL) of HABA-Gly-OPfp (37 mg, 80 μmol) and ^(Boc)PNA-OH (18mg, 80 μmol) and stirred at room temperature for 15 hours. This wasconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (0-30% MeOH/CH₂Cl₂) to giveHABA-Gly-^(Boc)PNA-OH (13 mg, 33%). ¹H NMR (CDCl₃) δ 9.77 (s) and 9.59(s) (1 H), 8.26 (s) and 8.14 (s) (2 H) ; 7.9-7.6 (m, 2 H), 7.6-7.3 (m, 2H), 7.0-6.6 (m, 2 H), 5.35 (s) and 5.05 (s) (1 H), 4.40 (s) and 4.24 (s)(2 H), 3.98 (s, 2 H) 3.6-3.3 (m, 2 H), 3.21 (s) and 3.02 (s) (2 H), 1.28(s) and 1.18 (s) (9 H).

Example 18 Synthesis of 2,3,4,5,6-pentafluorophenyl2-(5,7,8-trimethyl-1,3-dioxo-2,5-dihydro-2,4-diazaphenazin-2-yl)acetate(Flavin-Opfp)

[0087] EDC (73.2 mg, 382 μmol) was added to a DMF solution (10 mL) ofFlavin (100 mg, 318 μmol) and PfpOH (70.2 mg, 381 μmol) at 0° C., andthe reaction mixture was stirred at 0° C. for 1 hour and at roomtemperature for 12 hours. The reaction mixture was concentrated underreduced pressure, and the residue was subjected to partition extractionwith a water-chloroform system. The organic layer was dried withmagnesium sulphate and concentrated under reduced pressure, and theresidue was purified by silica gel column chromatography (2.5%MeOH/CHCl₃) to give Flavin-OPfp (130 mg, 85%). ¹H NMR (CDCl₃) δ 8.07 (s,1 H), 7.44 (s, 1 H), 5.21 (s, 2 H), 4.14 (s, 3 H), 2.55 (s, 3 H), 2.45(s, 3 H); HRMS (FAB⁺, NBA/CH₂Cl₂) calcd. for C₂₁H₁₄O₄N₄F₅ [(M+H)⁺]481.0934, observed 481.0950; UV λmax (DMF) 390, 460 (nm)

Example 19 Synthesis of2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-2-(5,7,8-trimethyl-1,3-dioxo(2,5-dihydro-2,4-di-azaphenazin-2-yl)acetylamino)aceticacid (Flavin-^(Boc)PNA-OH)

[0088] Diisopropylethylamine (36.3 μL, 208 μmol) was added to a DMFsolution (10 mL) of Flavin-OPfp (100 mg, 208 μmol) and ^(Boc)PNA-OH(45.4 mg, 208 μmol) and stirred at room temperature for 15 hours. Thiswas concentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (10-50% MeOH/CHCl₃) to giveFlavin-^(Boc)PNA-OH (130 mg, 85%). ¹H NMR (CD₃OD) δ 7.94 (s) and 7.86(s) (1 H), 7.80 (s) and 7.75 (s) (1 H), 5.03 (s) and 4.88 (s) (2 H),4.17 (s) and 4.13 (s) (3 H), 3.64 and 3.52 (2 H), 3.38 and 3.26 (2 H),2.58 (s) and 2.56 (s) (3 H), 2.46 (s) and 2.44 (s) (3 H), 1.46 (s) and1.41 (s) (9 H); HRMS (FAB⁺, NBA/CH₂Cl₂) calcd. for C₂₄H₃₁O₇N₆ [(M+H)⁺]515.2252, observed 515.2273; UV λmax (DMF) 390, 460 (nm).

Example 20 Synthesis of 2′,3′,4′,5′,6′-pentafluorophenyl1,3-dioxo-1H-benz[de]isoquinoline-2 (3H)-acetate (NI—Opfp)

[0089] DCC (155 mg, 0.75 mmol) was added to a DMF solution (5 mL) ofNI—OH (192 mg, 0.75 mmol) and PfpOH (152 mg, 0.83 mmol) while icecooling, and the reaction mixture was stirred at room temperature for 15hours. The reaction mixture was filtered, the filtrate was concentratedunder reduced pressure, and the residue was purified by silica gelcolumn chromatography (CH₂Cl₂) to give NI—OPfp (277 mg, 87%) as a redpowder. ¹H NMR (CDCl₃) δ 8.64 (d, J=8 Hz, 2 H), 8.25 (d, J=8 Hz, 2 H),7.78 (t, J=8 Hz, 2 H), 5.29 (s, 2 H).

Example 21 Synthesisof2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-2-(1,3-dioxo-1H-benz[de]isoquinoline-2(3H))acetylamino)aceticacid (NI-^(Boc)PNA-OH)

[0090] Diisopropylethylamine (87 μL, 0.50 mmol) was added to a DMFsolution (10 mL) of NI—OPfp (211 mg, 0.50 mmol) and ^(Boc)PNA-OH (120mg, 0.55 mmol) and stirred at room temperature for 15 hours. This wasconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (0-20% MeOH/CHCl₃) to giveNI-^(Boc)PNA-OH (130 mg, 85%). ¹H NMR (DMSO-d₆) δ 8.47 (m, 4 H), 7.86(dd, J=8.3, 7.3 Hz, 2 H), 4.73 (s, 2 H); ¹³C NMR (CDCl₃) δ 167.84,163.59, 134.20, 131.44, 131.39, 128.11, 126.78, 122.00, 61.59, 41.44,14.29; HRMS (FAB⁺, NBA/CH₂Cl₂) calculated for (C₁₆H₁₃NO₄)H⁺ 284.2933,observed 456.1767; UV λmax (DMF) 333 nm.

Example 22 Synthesis of 2′,3′,4′,5′,6′-pentafluorophenyl1,3-dioxo-5-nitro-1H-benz[de]isoquinoline-2(3H) -acetate (NI(NO₂)—Opfp)

[0091] EDC (73.2 mg, 382 μmol) was added to a DMF solution (10 mL) ofNI(NO₂)—OH (100 mg, 318 μmol) and PfpOH (70.2 mg, 381 μmol) at 0° C.,and the reaction mixture was stirred at 0° C. for 1 hour and at roomtemperature for 12 hours. The reaction mixture was concentrated underreduced pressure, and the residue was subjected to partition extractionwith a water-chloroform system. The organic layer was dried withmagnesium sulphate and concentrated under reduced pressure, and theresidue was purified by silica gel column chromatography (2.5%MeOH/CHCl₃) to give NI(NO₂)—OPfp (130 mg, 85%). ¹H NMR (CDCl₃) δ 8.07(s, 1 H), 7.44 (s, 1 H), 5.21 (s, 2 H), 4.14 (s, 3 H), 2.55 (s, 3 H),2.45 (s, 3 H); HRMS (FAB⁺, NBA/CH₂Cl₂) calcd. for C₂₁H₁₄O₄N₄F₅ [(M+H)⁺]481.0934, observed 481.0950; UV λmax (DMF) 390, 460 (nm).

Example 23 Synthesis of2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-2-(1,3-dioxo-5-nitro-1H-benz[de]isoquinoline-2(3H))acetylamino)aceticacid (NI(NO₂)-^(Boc)PNA-OH)

[0092] Diisopropylethylamine (36.3 μL, 208 μmol) was added to a DMFsolution (10 mL) of NI(NO₂)—OPfp (100 mg, 208 μmol) and ^(Boc)PNA-OH(45.4 mg, 208 μmol) and stirred at room temperature for 15 hours. Thiswas concentrated under reduced pressure and the residue was purified bysilica gel column chromatography (10-50% MeOH/CHCl₃) to giveNI(NO₂)-^(Boc)PNA-OH (130 mg, 85%). ¹H NMR (CD₃OD) δ 7.94 (s) and 7.86(s) (1 H), 7.80 (s) and 7.75 (s) (1 H), 5.03 (s) and 4.88 (s) (2 H),4.17 (s) and 4.13 (s) (3 H), 3.64 and 3.52 (2 H), 3.38. and 3.26 (2 H),2.58 (s) and 2.56 (s) (3 H), 2.46 (s) and 2.44 (s) (3 H), 1.46 (s) and1.41 (s) (9 H); HRMS (FAB⁺, NBA/CH₂Cl₂) calcd. for C₂₄H₃₁O₇N₆ [(M+H)⁺]515.2252, observed 515.2273; UV λmax (DMF) 390, 460 (nm).

Example 24 Synthesis of5-acetylamino-1,3-dioxo-1H-benz[de]isoquinoline-2(3H)-acetic Acid(NI(NHAc)—OH)

[0093] NI(NH₂)—OH (100 mg, 0.37 mmol) was dissolved in pyridine (3 mL)and Ac₂O (3 mL) and stirred at room temperature for 15 hours. Themixture was concentrated under reduced pressure, then washed withdichloromethane, filtered and dried to give NI(NHAc)—OH (103.2 mg, 89%).¹H NMR (DMSO-d₆) δ 8.79 (s, 1 H), 8.61 (s, 1 H), 8.40 (d, J=8 Hz, 1 H),8.37 (d, J=8 Hz, 1 H), 7.82 (t, J=8 Hz, 1 H), 4.71 (s, 2 H), 2.16 (s, 3H).

Example 25 Synthesis of 2′,3′,4′,5′,6′-pentafluorophenyl5-acetylamino-1,3-dioxo-1H-benz[de]isoquinoline-2(3H)-acetate(NI(NHAc)—Opfp)

[0094] DCC (71 mg, 0.34 mmol) was added to a DMF solution (5 mL) ofNI(NHAc)—OH (97 mg, 0.31 mmol) and PfpOH (63 mg, 0.34 mmol) at 0° C. andthe reaction mixture was stirred at 0° C. for 1 hour and at roomtemperature for 15 hours. The reaction mixture was filtered and thenconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (0-20% acetone/CHCl₃) to giveNI(NHAc)—OPfp (140 mg, 95%). ¹H NMR (CDCl₃) δ 8.96 (s, 1 H), 8.53 (d,J=7.7 Hz, 1 H), 8.32 (d, J=1.6 Hz, 1 H), 8.20 (d, J=7.7 Hz, 1 H), (t,J=7.5 Hz, 2 H), 7.82 (brs, 1 H), 7.74 (d, J=7.7 Hz, 1 H), 5.27 (s, 2 H),2.28 (s, 3 H).

Example 26 Synthesis of2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-2-(5-acetylamino-1,3-dioxo-1H-benz[de]isoquinoline-2(3H))acetylamino)acetic acid (NI(NHAc)-^(Boc)PNA-OH)

[0095] Diisopropylethylamine (54 μL, 0.30 mmol) was added to a DMFsolution (5 mL) of NI(NHAc)—OPfp (140 mg, 0.29 mmol) and ^(Boc)PNA—OH(67 mg, 0.30 mmol) and stirred at room temperature for 15 hours. Thiswas concentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (2-20% MeOH/CHCl₃) to giveNI(NHAc)-^(Boc)PNA-OH (117 mg, 85%). ¹H NMR (DMSO-d₆) δ 8.4-7.3 (m, 5H), 5.05 (brs) and 4.90 (brs) (2 H), 3.76 (brs) and 3.54 (brs) (2 H),3.64 (s) and 3.49 (s) (2 H), 3.54 (brs) and 3.41 (brs) (2 H), 2.15 (s)and 2.04 (s) (3 H), 1.48 (s) and 1.45 (s) (9 H).

Example 27 Synthesis of succinimidylN-4-dimethylaminoazobenzene-3′-carbonate (m-MR—Osu)

[0096] DCC (100 mg, 0.50 mmol) was added to a DMF solution (7 mL) ofm-methyl Red (m-MR—OH; 110 mg, 0.41 mmol) and N-hydroxysuccinimide (60mg, 0.52 mmol) at 0° C., and the reaction mixture was stirred for 30minutes and then at room temperature for 15 hours. The reaction mixturewas filtered and distilled under reduced pressure, and the residue wassubjected to silica gel column chromatography (CH₂Cl₂) to give m-MR—OSu(124 mg; 82%) as an orange powder. ¹H NMR (CDCl₃) δ 8.59 (s, 1 H), 8.13(t, J=9.1 Hz, 2 H), 7.90 (d, J=9.1 Hz, 2 H), 7.61 (t, J=7.9 Hz, 1 H),6.77 (d, J=9.1 Hz, 2 H), 3.11 (s, 6 H), 2.93 (brs, 4 H)

Example 28 Synthesis of2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-2-(4-dimethylaminoazobenzene-3′-carbonylamino)acetylamino)aceticacid (m-MR-Gly-^(Boc)PNA-OH)

[0097] m-MR—OSu (73 mg, 0.20 mmol) and triethylamine (350 μL, 2.7 mmol)were added in that order to a DMF solution (10 mL) of Gly-^(Boc)PNA-OH(50 mg, 0.18 mmol) and stirred at room temperature for 15 hours. Aftercompletion of the reaction, the mixture was concentrated under reducedpressure, and the residue was subjected to silica gel columnchromatography (0-10% MeOH/dichloromethane) to givem-MR-Gly-^(Boc)PNA-OH (95 mg, 100%) as an orange powder. ¹H NMR(DMSO-d₆) δ 8.26 (s, 1 H), 7.92 (d, J=7.6 Hz, 2 H) 7.83 (d, J=9.1 Hz, 2H), 7.62 (t, J=7.6 Hz, 1 H), 6.88 (brt) and 6.74 (brt) (1 H), 6.85 (d,J=9.1 Hz, 2 H), 4.22 (d, J=2.7 Hz, 2 H); 3.99 (s) and 3.89 (s) (2 H),3.44 (t, J=6.4 Hz, 1 H), 3.4-3.25 (brs, 4 H), 3.07 (s, 6 H), 1.39 (s)and 1.37 (s) (9 H).

Example 29 Synthesis of2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-N′-((1-pyrenyl-n-butyl)glycyl))aceticacid (Pyrene-Gly-^(Boc)PNA-OH).

[0098] Pyrene-OSu (39 mg, 0.10 mmol) and triethylamine (138 μL, 1.0mmol) were added in that order to a DMF solution (5 mL) ofGly-^(Boc)PNA-OH (25 mg, 0.09 mmol) and stirred at room temperature for15 hours. After completion of the reaction, the mixture was concentratedunder reduced pressure, and the residue was subjected to silica gelcolumn chromatography (0-10% MeOH/dichloromethane) to givePyrene-Gly-^(Boc)PNA-OH (30 mg, 61%) as a pale yellow powder. HRMS(FAB⁺) calcd. for C₃₁H₃₅O₆N₃Na [(M+Na)⁺] 568.2526, observed 568.2429.

Example 30 Synthesis of2-(N-(2-((tert-butoxy)carbonylamino)ethyl)-2-(7-diethylaminocoumarin-3-carbonyl)glycyl)aceticacid (Coumarin-Gly-^(Boc)PNA-OH)

[0099] Coumarin-OSu (15 mg, 0.042 mmol) and triethylamine (55.5 μL, 0.4mmol) were added in that order to a DMF solution (5 mL) ofGly-^(Boc)PNA-OH (12.7 mg, 0.046 mmol) and stirred at room temperaturefor 15 hours. After completion of the reaction, the mixture wasconcentrated under reduced pressure, and the residue was subjected tosilica gel column chromatography (0-20% MeOH/dichloromethane) to giveCoumarin-Gly-^(Boc)PNA-OH (23 mg, 100%) as a yellow powder. ¹H NMR(DMSO-d₆) δ 8.68 (s) and 8.66 (s) (1 H), 7.70 and 7.69 (each d, J=9.1Hz) (1 H), 6.89 (brt) and 6.75 (brt) (1 H), 6.80 (d, J=9.1 Hz, 1 H),6.62 (s, 1 H), 4.25 (brd) and 4.07 (brd) (2 H), 4.13 (m, 1 H), 3.98 (s)and 3.89 (s) (2 H), 3.48 (q, J=6.8 Hz, 4 H), 3.35 (m, 2 H), 3.13 (brq)and 3.07 (brq) (2 H), 1.37 (s) and 1.36 (s) (9 H), 1.14 (t, J=6.8 Hz, 6H).

Industrial Applicability

[0100] The novel functional PNA monomers in accordance with the presentinvention can be applied to the assembly of PNA that is used in, forexample, gene therapy. Furthermore, in accordance with the presentinvention, a functional molecule can be efficiently introduced into PNAby two complementary synthetic routes B and C to the functional PNAmonomers. The present invention can therefore be applied industrially,for example to an industrial synthesis of a functional PNA monomer unitusing a Boc type monomer unit or a benzyloxycarbonyl-ω-amino acidderivative.

1. A compound represented by general formula (I) below:

(In the formula, A denotes

B denotes

R denotes H, NO₂, NH₂, NHCbz, Br, F, Cl or SO₃Na₂, and n is an integerof 1 to 4, wherein when A denotes

then B denotes

).
 2. A process for producing a functional PNA monomer by reacting at-butoxycarbonylaminoethylamine with a derivative of a functionalmolecule so as to incorporate the functional molecule into a PNAmonomer, characterised in that the derivative of a functional moleculeis an activated ester, wherein activated ester is characterised to have,on the carbonyl carbon forming the ester bond, a group represented bygeneral formula (II) below,

(In the formula, A denotes

R denotes H, NO₂, NH₂, NHCbz, Br, F, Cl or SO₃Na₂, and n is an integerof 1 to 4.), and, is characterised to have, on its carbonyl carbon, apentafluorophenoxy group or a succinimidoxy group.
 3. A process forproducing the activated ester according to claim 2, characterised inthat it comprises reacting a carboxylic acid derivative of a functionalmolecule with a compound having a pentafluorophenoxy group or asuccinimidoxy group.
 4. A process for producing the carboxylic acidderivative of a functional molecule according to claim 5, characterisedin that it comprises reacting a derivative of the functional moleculewith an aliphatic carboxylic acid.
 5. A process for producing afunctional PNA monomer by reacting a derivative of a functionalmolecule, including following chemical formula (IV) at the terminal ofmolecule, with an ω-amino acid derivative represented by general formula(III) below, thereby incorporating the functional molecule into a PNAmonomer, characterised in that the derivative of the functional moleculeis an activated ester:

(In the formula, R¹ denotes a hydrogen atom or a straight- orbranched-chain C₁ to C₅ alkyl group, and m denotes an integer of 1 to11.),


6. The production process according to claim 5, characterised in thatthe activated ester has, on its carbonyl group forming an ester bond, agroup represented by general formula (II) below, either directly or viaan aliphatic chain or a peptide chain:

(In the formula, A denotes

R denotes H, NO₂, NH₂, NHCbz, Br, F, Cl or SO₃Na₂, and n is an integerof 1 to 4.).
 7. The production process according to either claim 5 orclaim 6, characterised in that the activated ester has, on its carbonylcarbon, a pentafluorophenoxy group or a succinimidoxy group.